In vitro screening of EPS‐producing Streptococcus thermophilus strains for their probiotic potential from Dahi

Abstract Dahi is a very common and traditional fermented dairy product in Pakistan and its neighboring countries, it represents a rich source for the isolation of many new strains of lactic acid bacteria (LAB). The major objective of this study was to evaluate the probiotic potential of novel exopolysaccharide (EPS)‐producing strains of S. thermophilus isolated from Dahi, sold in the local markets of Rawalpindi and Islamabad, Pakistan. In this study, 32 isolates of S. thermophilus were initially isolated from Dahi and out of these, 10 identified strains were further screened for their EPS‐producing ability. Maximum EPS production was estimated for RIY strain (133.0 ± 0.06), followed by RIH4 strain (103.83 ± 0.76) and RIRT2 strain (95.77 ± 0.22), respectively. Thereafter, in vitro studies revealed that these newly identified EPS‐producing strains of S. thermophilus fulfilled the basic requirements for probiotic functions; including resistance to harsh conditions of GIT, good cell surface hydrophobicity, auto‐aggregation, and co‐aggregation, especially against L. monocytogenes. Finally, the safety assessment displayed that these strains were also sensitive to clinical antibiotics, including vancomycin. Thus, these selected EPS strains of S. thermophilus act as potential candidates for biostabilizers in the preparation of consumer‐friendly fermented probiotic milk products.

. Due to this capability EPSs can be used in fermented foods as a natural emulsifier or food-grade hydrocolloid (Ruhmann et al., 2015). In addition to these functional properties, this bacterium is reported to have many health benefits for animals and humans, and numerous strains of S. thermophilus have been known to possess probiotic properties. Probiotics are briefly defined by WHO/ FAO (2006) as "live micro-organisms which when administered in sufficient amounts (i.e., minimum 10 6 CFU gm/L) (Shah, 2007) give their consumer or host a specific health benefit." They have either a direct or indirect impact on the gastrointestinal tract; including immunemodulation, mitigation of diarrhea due to miscellaneous causes, and prophylaxis of gastrointestinal infections. Probiotics are also effective against various intestinal diseases such as Helicobacter pylori infections, colon cancer, and inflammatory bowel disease (Marteau et al., 2001;Tuncer & Tuncer, 2014), as well as for lactose intolerance, blood cholesterol, bacterial vaginosis, and atopic dermatitis (Shah, 2007).
Probiotic strains must possess some basic characteristics including survival in simulated gastrointestinal (GI) tract conditions, antibacterial activity, cell aggregation, and cell surface hydrophobicity or bacterial adhesion to hydrocarbons (BATH), as a measure of intestinal colonization ability against adhesion of enteropathogens (Monteagudo-Mera et al., 2019). Previously, the higher colonization was observed for high hydrophobicity (De Souza et al., 2019;Miljkovic et al., 2015Miljkovic et al., , 2019. Essentially, BATH is associated with the adherence of strains and aggregation is the clumping of cells associated with their persistence in the GI tract (Saito et al., 2019). These properties are also required for probiotic starter culture development (Guarner et al., 2005;Miljkovic et al., 2015Miljkovic et al., , 2019Vinderola & Reinheimer, 2003).
A commonly used domestic dairy product in Asian countries including Pakistan is "Dahi"; an indigenous yogurt containing a mixture of LAB strains with Lactobacillus bulgaricus and Streptococcus thermophilus as major microbiota. Previous studies have confirmed the presence of LAB, including Lactobacillus bulgaricus (Ali et al., 2019), Lactobacillus acidophilus (Farid et al., 2021), and S. thermophilus (Mahmood et al., 2013) in Dahi samples collected from the markets of Rawalpindi and Islamabad, Pakistan.
These microorganisms have antibacterial activity against pathogenic bacteria and prevent gastrointestinal infections in the consumers, presenting probiotic effects (Mahmood et al., 2013;Soomro & Masud, 2012). However, studies related to the isolation of EPSproducing strains of S. thermophilus from Dahi, with well-studied characteristics and probiotic features, are limited. Therefore, the aim of this study was to assess the probiotic potential of new EPS-producing strains of S. thermophilus, with biostabilizing effects, obtained from a Dahi source which can be used in consumer-friendly dairy products.

| MATERIAL S AND ME THODS
Dahi samples (n = 101) were collected from the local markets of Rawalpindi and Islamabad. These samples were collected under aseptic conditions and taken immediately to the laboratory for analysis.

| Isolation and identification of S. thermophilus
The selective medium M17 (CM0817) Oxoid England was used to recover isolates of S. thermophilus from Dahi samples. M17 agar medium (composed of 3.725g M17 broth, 1.5% Technological agar and 10% lactose) was prepared in 100ml distilled water according to the instructions of the manufacturer; pH was adjusted to 6.9 with 6N NaOH, mixed on a magnetic stirrer and sterilized in a digital autoclave at 121°C for 15 min. (Hirayama, Japan). The agar was then poured into sterilized Petri dishes and allowed to solidify. Inoculation of collected samples was performed by the streaking method, on M17 agar plates, and incubated at 37°C for 24-48 h. The obtained isolates were tested through Gram staining and only Gram (+) colonies were further screened based on their morphological and biochemical properties, according to Buchanan and Gibbons (1974)

| Exopolysaccharide production of strains
The identified and characterized S. thermophilus strains were further evaluated for having exopolysaccharide production ability.
Finally, mucoid or ropy (EPS producing) colonies of S. thermophilus were selected and further assessed for their EPS production.
In order to estimate EPS production, strains were inoculated (2%) in sterilized fermentation medium and incubated at 42°C for 24 h. 100ml of fermentation medium was prepared by adding 7ml of 11% skim milk (LP0031) Oxoid England, 3.0g nutrient agar (Oxoid, England), and 1.0g tryptone (Oxoid, England) in distilled water to make the volume up to 100ml, mixed on magnetic stirrer, and autoclaved at 121°C for 15 min.

| EPS isolation
Exopolysaccharides were isolated from the fermented medium according to the method described by Rimada and Abraham (2003), with slight modifications. The fermented sample (100ml) was taken and heated to boiling point (100°C) in a water bath for about 15 min. in order to remove proteins (to inactivate enzymes) and polysaccharides attached to the cell walls. After cooling to room temperature, the sample was centrifuged at 8,000 rpm for 10 min.
to remove the cells. 17ml of 85% trichloro-acetic acid (TCA) was then added to the sample (100 ml), cooled to 4°C, and centrifuged again at 8,000 rpm for 10 min. EPS concentration in the supernatant was increased by precipitation with cold ethanol (−20°C), with a 1:3 concentration and stored overnight at 4°C. The final precipitate, obtained by centrifugation at 8000 rpm for 10 min., was dissolved in distilled water (100ml) and stored at 4°C. The collected pellets of EPS were again suspended and filtered through a dialysis tube (molecular weight cut-off 8-14 KDa, Beijing Solarbio Science & Technology Co., Ltd., China). The dialysis was performed against water for 48h., with water removal after every 8th hour. For further quantity determination, the solutions were prepared according to the method of Xu et al. (2010).

| EPS quantification
EPS quantification was carried out according to the method of Dubois (Dubois et al., 1956) based on the phenol-sulfuric acid method, with slight modifications. Firstly, 5% of phenol red solution was prepared in the distilled water then 2ml of sample (EPS solution) and 1ml of phenol solution were mixed in the test tube. 5ml of concentrated sulfuric acid was added to the mixture and left for 10 min. Then, the mixture was shaken by vortex and incubated at 30ºC for 10 min. (until development of a yellow-orange color). The control was prepared by adding 400µl phenol solution in 400µl of distilled water. Afterward, the absorbance of samples was measured by spectrophotometer (UV-9200) at 490nm, and readings were compared with the control to measure total carbohydrate content. The amount of EPSs in each sample was interpreted by using glucose standard calibration line and expressed as mg/L glucose equivalent. Calibration line is prepared by glucose solution (1mg/ml) as standard, using 6 different proportions as defined by Feldmane et al. (2013) and Muigei et al. (2013).

| Evaluation of technological properties of EPS-producing strains
The strains identified as EPS producers were used to ferment milk in order to determine technological properties, such as titratable acidity, curdling time, flavor, body, and texture of the curd. Sensory attributes (flavor, body, and texture) were determined through sensory evaluation method and titratable acidity by titration method. All the experiments were conducted in triplicates.

| Antibacterial activity of S. thermophilus strains
For measuring antibacterial activity, pathogenic strains E. coli ATCC25922, S. aureus ATCC6538, P. aeruginosa ATCC25923, and L. monocytogenes ATCC 19,115 were obtained from the Department of Pathology, Pakistan Institute of Medical Science (PIMS) (Mahmood et al., 2013). The stocks of all the strains were maintained in 20% (v/v) glycerol and stored at −80°C.
For this purpose, the paper disc method was used, as described by Soomro and Masud (2012), with slight modifications. Sterilized paper discs of 6-mm diameter made of Whatman filter paper no. 1, were kept on nutrient agar plates having a target pathogenic strain, whereas discs carried an adsorbed aliquot (20µl) of cell-free supernatant. pH of the nutrient agar medium was adjusted to 7.2. To obtain a cell-free supernatant, freshly overnight-grown culture was attained in broth medium, and its pH was adjusted to 5.5 with 1 M NaOH. It was then centrifuged at 13,000 rpm for 10 min and the supernatant (cell-free) was collected to send through a syringe filter (0.2µm) to remove bacterial cells. For comparison with the control, Ampicillin disc (10µg) was used as a reference antibiotic. The concentration of the overnight-grown culture of indicator strains was adjusted according to 0.5 McFarland turbidity standard. The plates were then kept in an incubator for 24 h at 37°C. Resulting clear inhibition zones, formed around paper discs, were then measured for evaluation of antibacterial activity. Inhibition zones or spectra round discs were computed in diameter (mm).

| Bile salt resistance and acid tolerance test
Bile salt tolerance and acid tolerance of isolates was conducted by the methods of Hassanzadazar et al. (2012) and Singhal et al. (2010), with slight modifications. For the acid tolerance test, M17 broth medium with adjusted pH values (2 and 3) was used to create in vitro acidic conditions of the gastrointestinal tract. pH 2 and 3 were adjusted with 1N HCl while pH 6.9 was adjusted to serve as a control.
Overnight-grown culture of S. thermophilus strains (1%) were then inoculated to M17 broth and incubated at 37°C for 5h. Percentage acid tolerance was found by measuring optical density (O.D.) at 600 nm using following formula: For bile salt tolerance, a fresh overnight-grown culture of S. thermophilus strains (1%) was used for inoculation in M17 broth medium, supplemented with 0.3% and 1.5% bile salts (w/V), while M17 broth without bile salt (0%) supplementation was used as the control.
Samples were incubated at 37°C for 6 h. and optical density (O.D.) was measured at 600 nm to determine the bile tolerance percentage of strains using the following formula:

| Auto-aggregation assay
Auto-aggregation assay was performed in line with the method as outlined by Kaushik et al. (2009), with slight modifications. For this purpose, cell pellets from fresh growth of isolates were obtained by centrifugation (8000 rpm for 10 min.). The cell pellet was then washed and resuspended in 0.01 M phosphate buffer saline (PBS).
Initial cell concentration (initial absorbance) was adjusted according to 0.5 McFarland standard at 600 nm and then incubated at 37°C for 2h. After 2 h, the suspension was centrifuged to obtain the cell pellet and mixed with the respective broth of equal volume removed. The supernatant was used to measure its absorbance (final absorbance) while the broth was used as the control. The following formula was used for calculating the percentage of auto-aggregation capability.

| Bacterial adherence to hydrocarbons (BATH) test
The method used, with some modifications, for determining percentage of bacterial adherence or hydrophobicity of S. thermophilus strains, was described by Kaushik et al. (2009). For this purpose, three different hydrocarbons (xylene, n-hexadecane, and dichloromethane) were selected for measuring selected strains adherence percentage to these hydrocarbons. Briefly, fresh overnight-grown culture was centrifuged (at 8000 rpm for 10 min.) to obtain the cell pellet. The cell pellet was then washed and resuspended in 2.5ml 0.01 M phosphate urea magnesium (PUM) buffer. Initial absorbance of cell suspension was set to 0.7 at 600 nm and then 1 ml of any tested hydrocarbon (xylene, n-hexadecane, or dichloromethane) was added to the cell suspension. This suspension was then incubated at 37°C for 10 min. and vortexed (2 min.) to mix the two phases and again incubated at 37°C for 1 hr. After the incubation period, phases were separated and the aqueous phase was collected carefully to measure its absorbance at 600nm, using the following formula:

| Antibiotic susceptibility assay
The disc diffusion method (paper disc method) was used to determine the S. thermophilus strains' susceptibility to antibiotics, as defined by Pisano et al. (2014), with some modifications.
Antibiotics (10) were selected for this test.
In this method, a bacterial lawn was prepared on agar plates with the concentration adjusted according to 0.5 McFarland standard and antibiotic discs were kept on it. These plates were then incubated at 37°C for 24 h and after 24 h clear zones or zones of inhibition (ZoI) were measured in diameters (mm) and compared with the interpretative zone diameters (CLSI M100-S21, 2011).
The results were indicated as susceptible, moderately susceptible, or resistant.

| Statistical study of data
The resulting data were statistically examined using Statistical package (SPSS 16.0 version). For this purpose, completely randomized design (CRD) was used and for graphical representation of the data Microsoft Excel was used. ANOVA (two way) followed by Tukey's test was also applied for statistical differences with a level of significance = 0.05 (Han et al., 2016). and 32 as cocci Gram positive and catalase negative. All selected isolates resulted negative for motility and spore formation ability as also reported by Sharma (2014).

| Isolation and identification of S. thermophilus
In order to screen out S. thermophilus from 32 isolates of cocci, isolated Gram-positive and catalase-negative cocci were further differentiated on the basis of their growth at different temperatures and NaCl concentrations as well as their carbon dioxide gas production from glucose and confirmed through analytical profile index (API) test. Each experiment was conducted in triplicates and only promising isolates were further propagated for selection. The isolates which grew at 45°C but could not grow at even 2% NaCl concentration and did not produce carbon dioxide gas from glucose (homo-fermentative), were selected. Sharma (2014) (2007) and Mahmood et al., (2013). The PCR results confirmed the 10 selected strains as S. thermophilus (Ali et al., 2019;Kullen et al., 2000;Suhartatik et al., 2014).
3.2 | Exopolysaccharide production of selected strains of S. thermophilus 3.2.1 | Screening of ropy and mucoid strains to assess EPS-producing ability The 10 strains identified as S. thermophilus were tested for EPS production ability. For this purpose, initially their ropiness and mucoid nature was assessed through a visual observation method, that is, ropiness test. The strains which formed long ropy like structures when picked with a sterile inoculation wire loop, were considered as ropy strains. According to Gomez (2006) and Zivkovic et al. (2015), this phenotypic character can be associated with the production of exopolysaccharides on solid medium, however, exopolysaccharides can be capsular polysaccharides (CPS) or ropy polysaccharides (RPS).
Capsulation was determined through staining with crystal violet and subsequently rinsing with 20% copper sulphate solution. The results obtained are shown in Table 1 and it can be seen that all ten selected technique. According to Behare et al. (2010), the strains forming ropy polysaccharides were considered to be better than strains forming capsular EPS and, due to this, can be used in dairy industry as a biothickener.

| Exopolysaccharide isolation and quantification
EPSs produced by the tested strains (ropy or capsular) were further isolated and then quantified by the trichloroacetic acid method followed by precipitation through the cold ethanol method. A similar method was used by Han et al. (2016) for isolation and measuring the concentration of these polysaccharides.
The results obtained regarding EPS concentration are summarized in Table 1. It can be observed that different strains produced different amounts of extracellular polymers with a significant difference (p < .05) among all the tested strains. The selected strains were able to produce EPS in skim milk medium, from 19.67 to 133.0 mg/l. Stingele et al. (1996) that reported the presence in S. thermophilus SFI6 of epsM and epsA genes responsible of exopolysaccharides synthesis. Maximum EPS production was observed in RIY in skim milk medium (133.0 a ± 0.06) followed by RIH4 (103.83 b ± 0.76) while minimum EPS production was observed in RIRT (19.67 j ± 0.57).
This variation in the results of EPS production might be attributed to the reason that exopolysaccharides production is dependent on the strains, which might be associated with the gene encoding on chromosome for EPS formation. In the literature it was reported that the total yield of EPS produced by the lactic acid bacteria (LAB) depends on the composition of the medium and conditions in which the organisms grow (i.e., medium, temperature, and incubation time) (Cerning et al., 1990). Gamar et al. (1997) also reported that EPS production and yield were influenced by the carbon source and concentration. Consequently, those strains which produced maximum quantity of EPS have a potential to replace the usage of chemical stabilizers in the dairy industry.

| Technological screening-a comparison of EPS-producing strains
Technological properties including acidity, curdling time, body and texture of curd, and other sensory features are summarized in Table 2.
As shown, EPS production greatly affects sensory evaluation, body,

| Antibacterial activity of S. thermophilus strains
Our traditional fermented dairy product, Dahi, can be used as a source of probiotics because the microbial isolates included strains of S. thermophilus, which is identified as a probiotic bacteria (Bhowmik et al., 2009;Mahmood et al., 2013). In addition to the primary role of their milk acidification, these bacterial strains of S. thermophilus produce secondary metabolites such as antibacterial peptides and possess other probiotic features.
EPS-producing strains were firstly investigated to ascertain their possible antimicrobial activity against food pathogens before determining other probiotic properties. Four pathogenic strains were used for this purpose (as shown in Table 3), namely L. monocytogenes ATCC 19,115, E. coli ATCC25922, S. aureus ATCC6538, and P. aeruginosa ATCC25923 as also previously used by Mahmood et al. (2013).
Therefore, determination of the antibacterial activity of S. thermophilus strains against these indicator strains would be a novel character.
It is revealed from the results that all the ten tested strains gave variable results and showed a wide range of antimicrobial activity against different pathogenic/indicator strains, having more or less zone of inhibition against one pathogen or more. These differences in the inhibitory activities of tested strains against different indicator strains may be due to their genotype or environmental factors.
The results of antibacterial activity of cell-free supernatants, from S. thermophilus strains, are presented in

Acid tolerance
If a minimum amount of 10 6 log CFU (Nagpal et al., 2012) bacterial culture tolerates pH up to 2-3, it can be a potential candidate for a probiotic, as the initial pH of the stomach is 1.5 and it reaches up to pH 3-4 as food enters, which can remain for 4-5 h (Slavin, 2013).
Low pH tolerance or acid tolerance of S. thermophilus strains was measured in vitro at two pH levels (pH 2 and pH 3). Only six strains out of ten selected were found to be tolerant to acid at both pH levels (2 and 3). The maximum tolerance under acidic conditions was observed in RIY with a 69% survival after 5h of incubation at pH 3 and 25% at pH 2, followed by RIH4, showing 65% survival at pH 3 for 5h and 20% at pH 2 ( Figure 3). RIH4 is further followed by RIK with 62% survival at pH 3 and 19% survival at pH 2. Strain RIRT2 has 58% survival at pH 3 and 16% at pH 2 while strains RIRT and RIR1L showed almost similar survival rates at both pH levels with 52% survival at pH 3 and 15% at pH 2. Control strain gave survival percentages of 10% at pH2 and 47% at pH 3 as compared to other selected strains. Consequently, it can be said that pH 2 was more harmful for S. thermophilus than pH 3, however, the viability of cells declined during incubation. All six strains which remained viable at pH 3 had a survival rate of more than 50% and hence can be probable candidates as a probiotic culture (Liong & Shah, 2005).
Several studies have determined that S. thermophilus strains were unable to grow at low pH levels (Haller et al., 2001;Khalil, 2009;Mahmood et al., 2013;Maurad & Meriem, 2008); Tuncer and Tuncer (2014) reported that pH 1 was more lethal to S. thermophilus ST8.01 than pH 3, but during incubation at pH 3 viability of cells still declined and the percentage of inhibition was found to be more than 99.99%, whereas at pH 5 it was 95.43% and viability was retained. Another study by Mahmood et al. (2013)

TA B L E 3
Antibacterial activity of cellfree supernatants from S. thermophilus strains against different food pathogens was resistant to pH greater than 2 but nonresistant to 1.5. Some studies related to other probiotic bacteria also gave similar findings to our results. Maurad and Meriem (2008) reported that L. plantarum strains survived up to 6h after incubation at pH 2. According to Aswathy et al. (2008), LAB including Streptococcus growth increased at pH 5 and facilitated in the production of fermented vegetable and milk products.

Bile tolerance
Bile tolerance is one of the most essential criteria for a strain to be used as a probiotic culture (Hassanzadazar et al., 2012;Soleimanian-Zad et al., 2009;Vizoso-Pinto et al., 2006). Bile resistance and the ability of LAB to inhabit the intestinal tract appear to be correlated (Soomro & Masud, 2012). According to Aswathy et al. (2008), probiotic strains which are intended to be used for humans must have resistance to bile salts at 0.3% concentration.

| Cell aggregation
Auto-aggregation ability of probiotics is a prerequisite for their adherence with the epithelium cells of the intestine (Aslim et al., 2007;Collado et al., 2008). It can be seen from Figure 5 that the cellular aggregation percentage was variable for all the six selected strains. Maximum auto-aggregation was found for RIRT (98.8 ± 0.6) followed by RIY (97.8 ± 0.4), RIRT2 (61.2 ± 1.0), and RIH4 (53.6 ± 0.6), respectively, while the minimum was observed for RIR1L (12.0 ± 0.5) and RIK (8.8 ± 0.6). These variations can be probably due to the auto-aggregation ability of the single strain as also observed by other researchers (Kos et al., 2003;Todorov et al., 2009;Tuncer & Tuncer, 2014;Vlkova et al., 2008) reporting that physico-chemical properties of cell surfaces such as hydrophobicity might have affected the auto-aggregation abilities. The results in Table 4 show that high EPS-producing strains exhibited more aggregation. Aslim et al. (2007) and Darilmaz and Beyatli (2012) also reported that high EPS-producing strains exhibited significant aggregation. However, RIRT strain is less EPS producing but showed high auto-aggregation ability which might be due to the strain specificity.

| Bacterial adherence to hydrocarbons (BATH)
The ability of bacteria to adhere to different hydrocarbons is the measure of the bacterial hydrophobicity to assess adherence of bacterial strains to the intestinal lining. Previously, in vitro analysis of bacterial adhesion to hydrocarbons using n-hexadecane and xylene was carried out by Schillinger et al. (2005) and Kaushik et al. (2009), while using dichloromethane as a source of hydrocarbons was conducted by Jose et al. (2015).
In the present study, three hydrocarbons were used, namely nhexadecane, xylene, and dichloromethane (DCM) for testing the adherence percentage of selected strains of S. thermophilus, as shown in Figure 6. Among the three different hydrocarbons used, there is significant difference (p <.05) between n-hexadecane and the other two hydrocarbons (xylene and dichloromethane), whereas between xylene and dichloromethane there is a nonsignificant difference (p >.05). In contrast to this study, Kaushik et al. (2009) Iyer et al. (2010). Although small differences exist for adherence percentage, the present study values are still higher than many other findings ( Figure 6). According to the criterion as described by Tyfa et al. (2015)

| Antibiotic susceptibility
A key requirement for probiotic strains is that they should not carry transmissible antibiotic resistance genes. Ingestion of bacteria carrying such genes is undesirable, as horizontal gene transfer to recipient bacteria in the gut could lead to the development of new antibiotic-resistant pathogens (Guglielmetti et al., 2009;Saarela et al., 2000;Salminen et al., 1998). For this, the assessment of S. thermophilus strains' susceptibility to clinically important antibiotics becomes important (Tuncer & Tuncer, 2014). The six selected strains were tested against 10 antibiotics by agar diffusion method as presented in Table 4. These strains were grouped as susceptible (S:20mm or >), resistant (R:0-10mm), or intermedi-  Temmerman et al. (2003), Aslim and Beyatli, (2004), Tosi et al. (2007), and Mahmood et al. (2013). These differences in the degree of inhibition with various antibiotics were possibly due to the difference in environment of strain isolation, as this is not the intrinsic feature of strains, or might be due to their different actions on the cell components such as the cell wall, protein and DNA synthesis, DNA gyrase, and RNA polymerase (Neu, 1992).

| CON CLUS ION
Today, the selection of probiotic, natural, EPS-producing strains is gaining importance throughout the world for replacing artificial stabilizers. The present in vitro findings reflected that these three novel EPS-producing strains of S. thermophilus (RIRT2, RIH4, and RIY), isolated from indigenous Dahi samples fulfill the basic criteria for the selection of probiotics with additional health benefits.
Thus, these strains have a potential to be used as a source of biostabilizer starter culture for the different probiotic fermented milk products.

ACK N OWLED G M ENTS
The authors thank the Institute of Food and Nutritional Sciences, F I G U R E 6 Adherence to different hydrocarbons of S. thermophilus strains isolated from local Dahi (mean ± SD)

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available